Vaccine comprising recombinant ct or lt toxin

ABSTRACT

The present invention provides a recombinant toxin or the subunit B thereof selected from the group consisting of  E. coli  heat-labile enterotoxin (LT), its subunit B (LTB), cholera toxin (CT) and its subunit B (CTB), in immunogenic form, expressed in eukaryotic cells, a vaccine comprising said toxin or subunit B thereof, and use of said recombinant toxin or subunit B thereof in human or veterinary vaccines.

FIELD OF THE INVENTION

The present invention relates to the production in eukaryotic cells ofrecombinant cholera toxin (CT) and E. coli enterotoxin (LT) and their Bsubunits CTB and LTB, respectively, and to their use as vaccines or asadjuvants in vaccines with various antigens.

ABBREVIATIONS: AOX1: alcohol oxidase I; AOX2: alcohol oxidase II; BMGY:buffered glycerol-complex medium; BMMY: buffered methanol-complexmedium; CHO: Chinese hamster ovary; CMV: cytomegalovirus; CT: choleratoxin of Vibrio cholera; CTA: cholera toxin of Vibrio cholera subunit A;CTB: cholera toxin of Vibrio cholera subunit B; ETEC: enterotoxigenic E.coli; HF cells: high five cells; HRP: horseradish peroxidase; IBDV:infectious bursal disease virus; LT: heat-labile enterotoxin ofEscherichia coli; LTA: heat-labile enterotoxin of Escherichia colisubunit A; LTB: heat-labile enterotoxin of Escherichia coli subunit B;MM: Minimal Methanol; rLTB: recombinant LTB; VP2: Viral protein 2;yrLTB: yeast rLTB.

BACKGROUND OF THE INVENTION

Vaccination is the main method of protecting humans and animals againstinfectious diseases. In response to active vaccination, antibodies andmemory B or T cells are produced which confer protection for longperiods (on the order of years). Vaccines consist of the live,attenuated pathogen, the inactivated pathogen, or components of thepathogen. In the present era of genetic engineering, subunit vaccinesare also being used, usually by producing a polypeptide of the pathogenin an expression system. Neutralizing antibodies to such a vaccine areinduced upon injection of animals with an adjuvant (Liu, 1998).

The heat-labile enterotoxin of Escherichia coli (LT) and cholera toxinof Vibrio cholera (CT) cause two very serious diseases in developingcountries. Both have similar pathogenic effects and show 95% sequencesimilarity (De Haan et al., 1999; Foss and Murtaugh, 1999), raising thepossibility of using the LT or its subunit B molecule for vaccinationagainst cholera. However, this molecule needs to be engineered in orderto prevent the damage incurred by exposure to wild-type LT. Both toxinsconsist of five non-toxic B (CTB, LTB) subunits and one toxic A subunit,with a loop that is a central target for biological manipulation(Yamamoto and Yokota, 1983; Sixma et al., 1991; Yamamoto et al., 1984).The logical approach is therefore to use the non-toxic form instead ofthe native toxin.

Production of CTB or LTB without the A subunit has been attempted. CTBand LTB have been cloned and expressed in different expression systems,such as E. coli (L'Hoir et al., 1990; De Geus et al., 1997; Slos et al.,1994), Mycobacterium bovis (Hayward et al., 1999) and Lactobacillus orBacillus brevis (Slos et al., 1998; Isaka et al., 1999; Goto et al.,2000), or surface-displaced on Staphylococcus xylosus and S. carnosus(Liljeqvist et al., 1997).

In recently published research, the CTB subunit was cloned into an E.coli host cell and anti-CT antibodies recognized the expressed protein.Moreover, the recombinant protein damaged the cells in vivo (De Mattoset al., 2002).

When LTB is expressed in genetically engineered bacterial cells, theproduct needs to be purified from its endotoxins. However, chemicalpurification of LTB from wild-type E. coli or of CT expressed in V.cholerae cultures may leave traces of the holotoxin (De Mattos et al.,2002).

Another important aspect is the immunostimulatory function of theLT/LTB, CT/CTB molecules, and the use of these molecules as adjuvants invaccines (Ryan et al., 2001). This is based on LTB's potential to causeactivation and differentiation of immune system cells (Williams et al.,2000). CTB and LTB have bean found to be effective adjuvants inco-administration (Isaka et al., 1999) and genetic or chemical fusionwith antigens (Dertzbaugh et al., 1990).

LT and CT have been found to be effective mucosal adjuvants (De Haan etal., 1999; Walker et al., 1993; Rappuoli et al., 1999; Foss andMurtaugh, 1999; Liang et al., 1989; Ryan et al., 2001). LT and CT areboth secreted toxins with similar sequence structure and activity, whichcause diarrhea in humans (Spangler et al., 1992). LT is produced byenterotoxigenic E. coli (ETEC). Bacteria of this family produce twotypes of toxins, heat stable (ST) and heat labile (LT). The LT proteinis composed of two subunits: the subunit A (LTA), a 28-kDa polypeptide,confers LT's toxicity. The 60-kDa subunit B (LTB) is composed of fiveidentical polypeptides, which are synthesized separately with leaderpeptides for transfer to the cell periplasm. In the periplasm, theleader peptides are removed and a toxin unit is assembled bynon-covalent linkage between one LTA and five LTBs (AB5) (Yamamoto andYokota, 1983; Sixma et al., 1991; Spangler et al., 1992; Cheng et al.,2000; Yamamoto et al., 1984).

LTB, which has no toxic activity, is responsible for the binding of thetoxin. It binds mainly to cellular receptors, GM1 gangliosides, butalso, with lower affinity, to other gangliosides (Holmgren et al., 1985;Sugii and Tsuji, 1989; Spangler et al., 1992). Also CTB binds mainly tothe receptor, GM1 ganglioside, on the surface of susceptible cells, andmediate the entrance of the toxin into the cells, whereby the A subunit,upon proteolytic activation, causes diarrhea.

CT and LT are immunogenic molecules that stimulate systemic and mucosalimmune system responses (Hagiwar et al., 2001). However, the use of bothCT and LT as an adjuvant is limited, partly because of the toxicity ofCTA and LTA (Williams et al., 2000). Some studies have shown theimportance of ADP-ribosyl transferase in the adjuvant activity of LT(Lycke et al., 1992; Feil et al., 1996). In the last decade, strategieshave been developed to separate the adjuvant effect from the toxicity.Some researchers concluded that toxicity is part of the adjuvant effect,but others showed that the enzymatic effect of LTA is not essential forthis purpose (Dickinson, 1995; De Haan et al., 1999; Douce et al., 1995;Douce et al., 1997; Giuliani et al.; 1998; Hagiwar et al., 2001; Lu etal., 2002).

The mechanism that enables adjuvant activity has not been elucidated.However, one report has found that the immunogenicity and adjuvanteffects of LTB are dependent on its ability to bind to the cell receptor(most commonly GM1), whereas LT adjuvant characteristics are notdependent on binding to the receptor. This means that two independentmechanisms are involved in LT's enhancement of the immune response (deHaan et al., 1998; Ryan et al., 2001), and thus LTB can be used as anefficient carrier and adjuvant with no danger of toxification followingvaccination. LTB has been found to activate specific signals inlymphocytes that induce selective activation and differentiation ofthose cells (Williams et al., 2000). The binding of LTB to GM1 was foundto decrease the proliferation of mitogen-stimulated B cells on the onehand, and increase the expression of MHC class II and minorlymphocyte-stimulating determinants on the other (Francis et al., 1992).The effect of increasing MHC class II expression may explain theimmunostimulatory effect of LTB.

Inactivated vaccines are injected intramuscularly or subcutaneously.Since most pathogens enter via mucosal tissues, an effective localresponse in these systems may block the pathogen. In order to activatesuch an immune response, antigen must be transferred to the mucosa andtaken via dendritic cells to the peripheral lymph nodes, (McGhee et al.,1992; Boyaka et al., 1999; Ernst et al., 1999). Antibody level is themain parameter in such cases since this is the main way to neutralizetoxins or pathogens (Ryan et al., 2001). Intranasal vaccination with CTBadmixed with diphtheria toxoid elicits peripheral as well as systemicantibody responses (IgA and IgG, respectively) against the pathogen(Isaka et al., 1999). Similar results were found using LTB withinfluenza or bovine serum albumin (BSA) (Tochikubo et al., 1998).Moreover, following intranasal vaccination, antibodies were detected inother mucosal systems, such as the vagina (Verweij et al., 1998).

Addition of short polypeptides to the C-terminal or N-terminal end ofLTB does not interfere with its tertiary structure or biologicalactivity (Sandkvist et al., 1987; Schodel et al., 1989; Green et al.,1996; Sanchez et al., 1988; Dertzbaugh et al., 1990). Intranasalvaccination with peroxidase chemically linked to LTB induced a highlevel of anti-peroxidase antibodies in the sera, saliva, nasal fluidsand lungs (O'Dowd et al., 1999).

Viral protein 2 (VP2) of infectious bursal disease virus (IBDV) ofchicken has been found to induce the production of neutralizingantibodies when produced in a eukaryotic expression system (Pitcovski etal., 1996). This subunit vaccine was chosen as a model to show thepotential of yeast-produced LTB for use as an adjuvant and carrier ofsubunit vaccines.

SUMMARY OF THE INVENTION

It is the main object of the present invention to produce a recombinantprotein selected from CT, LT, CTB, and LTB in eukaryotic cells, therebyeliminating the toxicity of bacterial endotoxins, while retaining theadjuvant effect of CT, LT, CTB and LTB for neutralizing antibodiesinduced by the recombinant toxin or subunit thereof.

The present invention thus provides a recombinant toxin or the subunit Bthereof selected from the group consisting of E. coli heat-labileenterotoxin (LT), its subunit B (LTB), cholera toxin (CT) and itssubunit B (CTB), in immunogenic form, wherein said immunogenic toxin orthe subunit B thereof has been expressed in eukaryotic cells. In onepreferred embodiment, the eukaryotic cells are yeast cells, morepreferably, Pichia pastoris cells.

The recombinant toxins and subunits thereof can be used as vaccinesagainst the respective bacteria or as adjuvants in vaccines with variousantigens.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows LTB DNA fragment amplified by PCR. Lane 1: Molecular sizemarkers; lane 2: LTB (310 bp).

FIG. 2 shows screening of Pichia pastoris colonies expressingrecombinant LTB (rLTB) with specific anti-CT antibodies. 1-40—coloniestransformed with LTB. 50, 51—colonies transformed with wild-type plasmid(negative control).

FIGS. 3A-3B show identification of rLTB expression in yeast by SDS-PAGE(A) or Western blotting (B). FIG. 3A: SDS-PAGE of induction mediumstained with Coomassie blue to test expression of recombinant proteins.FIG. 3B: Immunoblot with anti-CT antibodies to detect rLTB proteinexpression during the induction. Lane 1: Commercial CTB protein; Lanes2, 3, 4: Supernatant of yeast with wild-type plasmid at 5, 6, 7 days ofinduction, respectively; Lanes 5, 6, 7: Supernatant of yeast expressingrLTB at 5, 6, 7 days of induction, respectively. Lane 8: Molecular sizemarker. Samples were loaded on gel without boiling to avoid reduction ofthe pentamer structure into monomers.

FIG. 4 shows dot blot to test expression levels of rLTB in response tomethanol concentration. Dots 1, 2, 3: rLTB expression followinginduction with 0.3%, 0.6% and 1.5% methanol, respectively; dots 4, 5:negative control—induction medium of wild-type transformed colonyfollowing induction with 0.3% or 1.5% methanol, respectively.

FIGS. 5A-5B are graphs showing rLTB protein purification by cationexchange chromatography. FIG. 5A: separation of induction medium of rLTBexpressed in yeast. FIG. 5B: separation of induction medium wild-typeplasmid expressed in yeast (negative control).

FIGS. 6A-6B: show purification of yeast rLTB (yrLTB) by cation-exchangechromatography tested by SDS-PAGE (A) and Western blotting (B). FIG. 6A:SDS-PAGE stained by Coomassie blue to test purification of yrLTB. FIG.6B: Immunoblot with anti-CT antibodies to test purification of yrLTB.Lane 1: commercial CT protein; lanes 2, 3: elution fraction (38% NaCl)of yrLTB and wt plasmid respectively; lanes 4, 5: elution fraction (41%NaCl) of yrLTB and wt plasmid respectively; lanes 6, 7: fractions 2, 3after boiling, respectively; lanes 8, 9: fractions 4, 5 after boiling,respectively. Broad arrow-pentamer of yrLTB protein. *-monomer of boiledyrLTB protein.

FIG. 7 shows DNA LTB-linker and VP2-linker fragments amplified byPCR—two first steps. Lane 1: molecular size markers; lane 2: LTB-linker(330 bp); lane 3: molecular size markers; lane 4: VP2-linker (1.42 kbp).

FIG. 8 shows LTB-VP2 DNA fragment amplified by PCR. Lane 1: molecularsize markers; lane 2: LTB-VP2 (1725 bp).

FIGS. 9A-9B show identification of LTB-VP2 expression in yeast bySDS-PAGE (A) or immunoblot (B). FIG. 9A: lane 1: supernatant fraction ofyeast with wild-type plasmid; lane 2: molecular size markers; lane 3:supernatant fraction of yeast expressing LTB-VP2. FIG. 9B: lane 1:supernatant fraction of yeast expressing LTB-VP2 detected by anti-CTantibodies; lane 2: supernatant fraction of yeast with wild type plasmiddetected by anti-CT antibodies; lane 3: boiled supernatant fraction ofyeast expressing LTB-VP2 detected by anti-CT antibodies; lane 4: boiledsupernatant fraction of yeast with wild-type plasmid detected by anti-CTantibodies. *-monomer of LTB-VP2 (46 kDa); broad arrow—pentamer ofLTB-VP2 (230 kDa).

FIG. 10 is an immunoblot to test for anti-CT antibodies in response tovaccination of broilers with rLTB expressed in yeast. The antigen CT(Sigma) was exposed to sera of birds vaccinated by: lane 1: commercialCT given orally; lane 2: induction medium of a colony expressing rLTB,given orally; lane 3: induction medium of a colony carrying wild-typeplasmid, given orally; lane 4: commercial CT given by injection; lane 5:induction medium of colony expressing rLTB given by injection; lane 6:induction medium of colony carrying wild-type plasmid given byinjection.

FIG. 11 is a graph showing anti-CT antibodies in broilers three weeksafter vaccination with rLTB expressed in yeast, as determined by ELISA.Statistically significant differences (P<0.05) are indicated by anasterisk (*).

FIG. 12 is a graph showing antibody response in chicks, vaccinated withrLTB at 1 day of age. Statistically significant differences (P<0.05) areindicated by an asterisk (*).

FIG. 13 is a graph showing anti-IBDV antibodies three weeks after secondvaccination with rLTB-VP2 expressed in yeast, as determined by ELISA.Statistically significant differences (P<0.05) are indicated by anasterisk (*).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one aspect, a recombinant toxin orthe subunit B thereof selected from the group consisting of E. coliheat-labile enterotoxin (LT), its subunit B (LTB), cholera toxin (CT)and its subunit B (CTB), in immunogenic form, wherein said immunogenictoxin or the subunit B thereof has been expressed in eukaryotic cells.

For the sake of clarity, it is to be understood that the term“recombinant CT, LT, CTB or LTB”, as used herein, refers to recombinantCT, LT, CTB or LTB produced in eukaryotic cells.

Expression in eukaryotic systems, as opposed to prokaryotes, has threemain advantages. The product is free of endotoxins, it is inexpensiveand it allows production of fusion proteins that requirepost-translational modifications (e.g. glycosylation andphosphorylation) in order to be immunogenic and elicit the production ofneutralizing antibodies. The resultant molecule may be used as a vaccineagainst the toxin itself or serve as an adjuvant in other vaccine.

According to one embodiment of the present invention, the eukaryoticsystem used is a yeast expression system. Yeast offer advantages overbacteria in heterologous protein production because, although they areunicellular organisms easy to manipulate and grow quickly, theircellular organization is eukaryotic, making it possible to performexpression and maturation processes characteristic of animal and plantcells. Moreover they can secrete recombinant proteins into the culturemedium, being recombinant product levels higher there than in thecytoplasm. Even more, the secreted products are obtained with a highdegree of purity (since few endogenous proteins are secreted) andtherefore the purification steps are reduced. Finally, they offer asuitable environment for the adequate folding of proteins, especially ofthose that contain disulfide bonds.

In a preferred embodiment of the present invention, the methylotrophicyeast Pichia pastoris (P. pastoris) is used as the expression system. P.pastoris is a yeast that can metabolize methanol as the sole source ofcarbon and energy (methylotrophic) and is currently used for theproduction of recombinant proteins since, as a production system, it issimpler, cheaper and more productive than other higher eukaryoticsystems. Being a yeast, it shares the advantages of easy genetic andbiochemical manipulation of Saccharomyces cerevisiae but surpasses itsheterologous protein production levels (10 to 100 times greater).According to the present invention, the CT, LT, CTB or LTB polypeptidecan be produced by P. pastoris cells whose genome contains at least onecopy of the cDNA sequence encoding said polypeptide, and under theregulation of a promoter region of a methylotrophic yeast gene that canbe induced with methanol. The polypeptide product produced according tothe present invention may be secreted into the culture medium in a highconcentration.

In another embodiment of the present invention, CT, LT, CTB or LTB isexpressed in mammalian cells. The recombinant DNA fragments encoding LT,CT, CTB or LTB are cloned into eukaryotic expression plasmids andtransfected into mammalian cells for stable or transient expression.According to the invention, the preferred mammalian cells are Chinesehamster ovary (CHO) cells.

In a further embodiment of the present invention, LT, CT, CTB or LTB areexpressed in insect cells through the baculovirus expression system.Recombinant baculoviruses are extensively used as vectors for abundantexpression of foreign proteins in insect cell cultures. The appeal ofthe system lies essentially in easy cloning techniques and viruspropagation combined with the eukaryotic post-translational modificationmachinery of the insect cell.

In preferred embodiments, the invention relates to the subunits B of LTand CT, and more preferably to LTB.

In another aspect, the invention provides a vaccine containing therecombinant LT, LTB, CT or CTB of the invention, more preferably LTB orCTB.

In one embodiment, the vaccine is a cholera vaccine containing therecombinant CT or CTB. In another embodiment, the vaccine is directedagainst E. coli heat-labile enterotoxin and contains the recombinant LTor LTB.

In a further aspect, the invention relates to the use of recombinant LT,LTB, CT or CTB, preferably produced in yeast cells, as an adjuvant inhuman or veterinary vaccines, and further provides a human or veterinaryvaccine comprising the recombinant LT, LTB, CT or CTB of the inventionand an antigen.

In one embodiment, the vaccine comprises a mixture of said recombinantLT, LTB, CT or CTB and said antigen. In another embodiment, the vaccinecomprises said recombinant LT, LTB, CT or CTB chemically linked to saidantigen. In a further embodiment, the vaccine comprises a fusion proteinformed by said recombinant LT, LTB, CT or CTB and said antigen. In stilla further embodiment, the said recombinant LT, LTB, CT or CTB can beco-administered with a human or veterinary vaccine.

The antigen for use in said vaccine of the invention may be any viral,bacterial, fungal or parasite antigen pathogenic to humans and/or toanimals such as, but not limited to, antigens related to hepatitis A, Bor C, or D virus, influenza virus, mouth and foot disease, cholera,rabies virus, herpes virus, human cytomegalovirus (CMV), dengue virus,respiratory syncytial virus, human papilloma virus, meningitis virus,Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Streptococcus,Mycoplasma, Mycobacteria, Haemophilus, Plasmodium or Toxoplasma,Pasteurella multocida, etc.

The vaccines of the invention are intended both for human and veterinaryuse, and may be for oral, intranasal, mucosal, eye drop, vaginal, rectaltranscutaneous or any other method of administration.

In one preferred embodiment, the invention provides a veterinary vaccinefor poultry vaccination against infectious bursal disease virus (IBDV)containing recombinant LT, LTB, CT or CTB, preferably produced in yeastcells, and the IBDV VP-2 antigen, more preferably, as a fusion protein.In a most preferred embodiment, the IBDV vaccine comprises recombinantLTB produced in Pichia pastoris and the IBDV VP-2 antigen, preferablywherein the LTB and the VP-2 moieties are linked by a linker peptide.Viral protein 2 (VP-2) of IBDV of chicken was found to induce theproduction of neutralizing antibodies when produced in a eukaryoticexpression system (Pitcovski et al, 1996). This subunit vaccine waschosen herein as a model to show the potential of yeast-produced LTB foruse as an adjuvant and carrier of subunit vaccines.

In one embodiment, the present invention further relates to arecombinant fusion protein comprising LT, LTB, CT or CTB and an antigenthat has to be expressed in eukaryotic cells, wherein said fusionprotein has been expressed in eukaryotic cells, preferably yeast, cells.In one preferred embodiment, said recombinant fusion protein isexpressed in the Pichia pastoris expression system.

In one embodiment, the recombinant fusion protein may comprise anantigen fused to LT via the B subunit of LT, or via the end of the Asubunit (A1 domain) of LT. Also, the recombinant fusion protein mayconsist of a fusion protein in which the antigen substitutes the A1domain of LT.

The antigen for use in said recombinant fusion protein may be any viral,bacterial, fungal or parasite antigen pathogenic to humans and/or toanimals such as, but not limited to, antigens related to hepatitis A, Bor C, or D virus, influenza virus, mouth and foot disease, cholera,rabies virus, herpes virus, human cytomegalovirus (CMV), dengue virus,respiratory syncytial virus, human papilloma virus, meningitis virus,Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Streptococcus,Mycoplasma, Mycobacteria, Haemophilus, Plasmodium or Toxoplasma,Pasteurella multocida, etc.

In yet another aspect, the invention provides an isolated DNA moleculecontaining one or more copies of an expression cassette that includes:

-   -   (i) an alcohol oxidase promoter of a methylotrophic Pichia        pastoris gene that can be induced with methanol;    -   (ii) a nucleotide sequence encoding LT, LTB, CT or CTB; and    -   (iii) an expression vector functional in Pichia pastoris.

The promoter region to be preferably used to lead the cDNA expressionencoding the LT, LTB, CT or CTB polypeptide is derived from the P.pastoris alcohol oxidase gene inducible with methanol. This yeast isknown to have two functional alcohol oxidase genes: alcohol oxidase I(AOX1) and alcohol oxidase II (AOX2). The coding regions of the two AOXgenes are closely homologous, their restriction maps are alike and theiramino acid sequence are very similar. The proteins expressed by the twogenes have similar enzymatic properties, but the AOX1 gene promoter ismore efficient with respect to its regulating function and rendershigher levels of the gene product than the AOX2 gene promoter; its useis therefore preferred for LT, LTB, CT or CTB expression according tothe present invention. The AOX1 gene, including its promoter, has beenisolated and reported in U.S. Pat. No. 4,855,231.

The invention further provides a Pichia pastoris yeast cell comprisingan expression vector that contains a nucleotide sequence encoding LT,LTB, CT or CTB, together with control elements enabling the expressionof said nucleotide sequence in yeast host cells. In one preferredembodiment, the Pichia pastoris cell is transformed by homologousrecombination with the DNA molecule above, particularly when thepromoter and the termination sequence are from the Pichia pastoris AOX1gene, wherein said DNA molecule integrates by homologous recombinationinto a Pichia pastoris which may use methanol as a sole carbon source.The transformed Pichia pastoris yeast cell may contain multiple copiesof the expression cassette.

In a further embodiment, the invention relates to a viable culture ofPichia pastoris cells containing the transformed cells, and to a processfor the production of a recombinant LT, LTB, CT or CTB polypeptidecomprising culturing the Pichia pastoris cell culture under conditionswherein said polypeptide is expressed and, if desired, secreted into theculture medium. Preferably, the culture is grown in a medium containingmethanol as a sole carbon source.

The invention further provides a recombinant LT, LTB, CT or CTB producedby the process as described herein above.

E. Coli enterotoxin (LT) and cholera toxin (CT) have been studiedintensively as vaccines against these diseases and as adjuvants formucosal vaccination. Two major problems interfere with the use of thesepromising molecules: their toxicity and the danger of other bacterialendotoxins being mixed in with the desired CT or LT. Expression of LTBor CTB by standard genetically engineered bacterial cells requiresfurther purification of the product from the bacterial endotoxins.However, chemical purification of LTB from wild-type E. coli or of CTexpressed in V. cholerae cultures may leave traces of the holotoxin (DeMattos, 2002).

The production of the recombinant toxins and, more particularly, of therecombinant B subunits CTB and LTB in eukaryotic cells according to theinvention, eliminates the undesired endotoxins and enables theproduction of large quantities of LTB or CTB.

In one most preferred embodiment of the present invention, rLTB wasexpressed in P. pastoris host cells as a biologically functionalprotein. This expression system has three main advantages over bacterialexpression systems. The first is that yeast cells do not produceendotoxins: because purification of endotoxins is an expensive processand it is hard to achieve totally pure samples, the use of yeast cellsmakes the purification process easier and the final product safer.Second, P. pastoris yeast cells are not pathogenic, even whenadministered live at very high concentrations (Pitcovski et al., 2003).Third, yeast is a eukaryotic organism that provides efficient and lessexpensive production of proteins as compared to expression in othereukaryotic systems. This system has been used for the production ofvarious recombinant proteins.

The ability to produce recombinant protein genetically conjugated to LTBin a eukaryotic system enables the use of LTB as an adjuvant in cases inwhich the antigen should be expressed in such a system due to the needfor glycosylation or other post-translational modifications. This wasthe case with the production of VP2, which provided protection only whenexpressed in a eukaryotic system. Another advantage of the yeast systemis that the protein is secreted into the medium. The purification issimple and the fusion protein is in the correct form.

LTs have been found to be similar in sequence, immunological andphysiochemical characteristics in various types of E. coli. In thepresent invention, the plasmid coding for LT was isolated from E. coliH10407, a strain that causes diarrhea in humans and is geographicallywidespread (Inoue et al., 1993). The LTB DNA fragment of the correctsize (310 bp) (Sixma et al., 1991) was amplified by PCR and cloned intoyeast cells. For comparison, the same gene was cloned in an E. coliexpression system. High levels of pentameric protein were expressed in20% of the yeast colonies. The protein was observed in the yeast culturesupernatant, and identified by SDS-PAGE and immunoblotting with anti-CTantibodies. Moreover, yrLTB showed the natural biological activity oftoxic LT—binding to the GM 1 receptor, and this activity disappearedfollowing denaturation by boiling. Consequently, the LTB expressed in P.pastoris is probably in its correct native form. Since only thepentameric form of LTB can bind to the GM1 receptor (Liljeqvist et al.,1997), it may be assumed that yrLTB is correctly folded. This is crucialsince the immunogenicity of LTB subunits is based exclusively on theirability to bind ganglioside receptors (De Haan et al., 1998; Green etal., 1996).

During the 3 last days of induction, the amount of yrLTB produced didnot increase, but replacing the medium allowed its continuous expressionin large quantities (data not shown). This might enable efficientscale-up for batch production of yrLTB. In E. coli, recombinant LTB orCTB were obtained in inclusion bodies in our studies (data notpublished) and others' (De Mattos et al., 2002), but the expression percell was limited and the extraction complicated.

Most of the protein in the growth medium was the recombinant protein;however its concentration was relatively low. Cation-exchangechromatography enabled, in one step, purification and concentration ofthe yrLTB. Fusing a foreign polypeptide to yrLTB could result in changesin its folding. The current method was performed under native conditionsand was based on the isoelectric point of the recombinant protein,avoiding changes in protein folding during separation. The purifiedyrLTB was obtained at high concentrations and showed biological activitysimilar to that observed prior to being run through the column.

Antibodies play a major role in neutralizing bacterial toxins andpreventing adherence to surface receptors on host cells r-LTB was foundto be an immunogenic molecule. Injection of 3 μg rLTB elicited thesecretion of specific antibodies. Following oral administration of 3 μgof rLTB, the recombinant protein successfully traversed the digestivesystem and an effective systemic antibody response was detected. Forcomparison, the effective oral dose of BSA to activate an antibodyresponse against it is 25 mg per bird per day for six days (Klipper etal., 2000). In previous studies, a low amount of CT elicited an immuneresponse via oral administration. This was related to itshyper-immunostimulatory effect, its ability to withstand digestion byproteolytic enzymes and its ability to penetrate intestinal cells.Recombinant LTB appears to have similar qualities. This ability of yrLTBto cross the digestive tract raises the possibility of fusing a proteinto it and performing oral vaccinations. Another interesting finding isthat by the oral route, an immune response could be achieved at as earlyas 1 day of age, in contrast to intramuscular injection which did notinduce any antibody response at that age.

The ability to enhance the immune response against a protein that isfused to yrLTB was tested with VP2. This protein is used as a commercialsubunit vaccine in poultry and confers protection against IBD whenproduced correctly in eukaryotic organisms upon administration with oiladjuvant (Pitcovski et al., 2003). No antibodies against LTB were found,leading us to conclude that the LTB pentamer is surrounded by five VP2proteins, so anti-CT antibodies cannot bind. However, vaccination withrLTB-VP2 conferred full protection against challenge with virulent IBDV.In previous studies, injection of a protein without an adjuvant did notlead to the production of neutralizing antibodies at protection levels.

According to the present invention, we have seen that immunization byVP2 via eye-drops, without adjuvant, did not initiate antibodysecretion, whereas yrLTB as a fusion protein with VP2 yielded an immuneresponse to VP2 after only one vaccination. Thus, the results of thisexperiment proved the adjuvant effect of yrLTB. yrLTB enhanced antibodyproduction against some of the antigens that were co-administered byintramuscular injection. No antibodies were detected to aco-administered protein given orally (data not shown). Therefore, theadvantage of a fused molecule is that it may allow oral vaccination.

In a safety study in mice, it was found that intranasal administrationof CT may target neuronal tissue and may promote uptake of vaccineproteins into olfactory neurons in addition to nasal-associatedlymphoreticular tissues (van Ginkel et al., 2000). In other studies,mutant LT was found to be an effective and safe adjuvant for nasalimmunization vaccine (Hagiwar et al., 2001). Clinical safety of LTdelivered transcutaneously was tested in adult volunteers and thevaccine was found to be safe and effective (Guerena-Burgueno et al.,2002).

In summary, high levels of purified yrLTB were expressed in P. pastorisyeast cells and were secreted into the culture medium. The protein waspurified and concentrated and was found to bind to GM1 ganglioside. Whenadministered by injection or orally to birds, high anti-LTB antibodytiters were produced. It was further found to be efficient as anadjuvant. The adjuvant quality of yrLTB was proven by co-administrationwith, or fusion to, antigens. Thus, this efficiently produced andpurified molecule can safely be used for vaccination against the toxinitself or as a carrier for a foreign vaccine molecule.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Materials and Methods

(i) Cloning the LTB Gene in the Yeast Pichia pastoris

E. coli plasmid H10407 (Yamamoto and Yokota, 1983; Inoue et al., 1993)was used as a template to synthesize the LTB fragment. The DNA sequenceof the fragment encoding the B subunit of LT was propagated by PCR usingprimers of the ends of the gene as published previously (GenBankaccession number AB011677). Sequence of primers: 5′ primer for the LTBconstruct: 5′ ccg ctc gag aag ctc ccc agt cta tta cag 3′ (SEQ ID NO: 1),3′ primer for the LTB construct: 5′ cgc gga tcc cta gtt ttc cat act gattgc cgc 3′ (SEQ ID NO: 2).

The reaction solution contained 1 unit of Taq polymerase (Promega,Madison, Wis., USA), 5 μl of Taq buffer (20 mM Tris-HCl pH 8.3, 50 mMKCl, 2 mM MgCl₂) and 20 pmol of each primer in a final volume of 50 μl.The PCR scheme was as follows: 5 min at 94° C., 2 min at 42° C., 60 s at74° C., (60 s at 94° C., 2 min at 50° C., 3 min at 74° C.)×28 cycles, 15min at 74° C. The PCR product was electrophoresed in a 1% agarose geland visualized by ethidium bromide staining.

The DNA fragment was purified with a “PCR products purification system”kit (GibcoBRL, UK) and cloned into a pHILSI plasmid (Invitrogen, SanDiego, Calif., USA) using the XhoI and BamHI restriction sitesincorporated into the primers. The recombinant plasmid was transformedinto Top10 E. coli cells (Invitrogen) according to the manufacturer'sinstructions.

The colonies that grew on LB plates containing ampicillin (100 μg/ml)were screened for the LTB fragment by PCR. The plasmid from the positivecolonies was purified by mini-preps kit (Promega) according tomanufacturer's instructions, and tested again with restriction enzymes.The DNA sequence of colonies carrying the desired gene was determined(Hebrew University, Biotechnology Services, Jerusalem, Israel).

To promote integration into the AOXI locus of the yeast genome, therecombinant plasmid was linearized with BglII restriction enzyme. Thelinearized r-plasmid was cloned into P. pastoris SMD1168 (Invitrogen)according to kit instructions as recommended by the manufacturer in: ‘AManual of Methods for Expression of Recombinant Proteins in Pichiapastoris’, Version 1.8.

(ii) Screening for Pichia pastoris Colonies Expressing LTB

All growth media and plates were prepared according to themanufacturer's recommendations (Invitrogen).

The recombinant colonies were grown on Regeneration Dextrose Base platesfor 4 days at 30° C. A hybond-C nitrocellulose membrane (AmershamInternational, Little Chalfont, UK) was placed over the Minimal Dextrose(MD) plate and single colonies were pasted onto the membrane. A colonytransfected with a wild-type plasmid was grown as a negative control.

After 2 days at 30° C., the membrane was transferred onto a MinimalMethanol (MM) plate. Following 7 days' incubation at 30° C., 100 μlmethanol was added twice a day to induce protein production. The samecolonies were grown and kept as master stocks.

The membrane was washed three times, 10 min each, in TNT buffer (150 mMNaCl, 10 mM Na₂HPO₄, 10 ml Tris-HCl pH 8.0, 0.05%, v/v, Tween 20) andblocked with milk buffer (150 mM NaCl, 10 mM Na₂HPO₄, 10 mM Tris-HCl pH7.0 in milk, 1%, w/v, fat) for 30 min. After two additional 10-minwashes in TNT buffer, the membrane was incubated with a 1:1000 dilutionof rabbit anti-CT polyclonal antibody (rabbit anti-CT, Sigma, St. Louis,Mo., USA) at 37° C. for 1 h. The membrane was washed twice with TNTbuffer and incubated with a secondary antibody—horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG (Sigma), under the sameconditions. Following two washes, the membrane was exposed to the HRPsubstrate solution 3,3′-diaminobenzidine (Sigma) until color developed.The stained colonies were isolated and stored at 4° C. for furthergrowth and expression.

(iii) Expression of Recombinant LTB in P. pastoris

All colonies that were detected by anti-CT were grown in 50 ml bufferedglycerol-complex medium (BMGY) at 30° C. for 48 h until the OD_(600nm)reached 10 to 20. The cells were centrifuged for 10 min at 6000 RPM andresuspended in 10 ml buffered methanol-complex medium (BMMY). At thispoint the expression of recombinant (r) LTB was induced by the additionof 300 μl methanol (to a final concentration of 0.3%, v/v) every 12 h.The optimal period of induction was determined by collecting samples oneach day of induction. The samples were centrifuged as previously. Thesupernatant, containing the soluble yeast (y) rLTB, was collected andstored at 4° C.

(iv) Protein and Antibody Assays—SDS-PAGE and Western Blotting rLTBprotein—The supernatant containing the soluble yeast rLTB was analyzedby SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot (Westernblot). Loading buffer (3%, w/v SDS and 5%, v/v mercaptoethanol) wasadded to each sample, which was then boiled, or not, and electrophoresedin 12% polyacrylamide slab gels, using a discontinuous SDS gel system(Bio Rad, Hercules, Calif., USA). In most cases, two slab gels wereelectrophoresed simultaneously. One was stained with Coomassie BrilliantBlue R, and the proteins from the second were electrotransferred onto anitrocellulose filter (Hybond C, Amersham International) using asemi-dry system (Bio Rad), and Western blotting was performed. Followingblocking with milk buffer, the membrane was incubated for 1 h at 37° C.with rabbit anti-CT antibody (Sigma) diluted 1:1000 in milk buffer.Filters were washed twice in PBS and incubated with goat anti-rabbitIgG-peroxidase conjugate (Sigma) diluted 1:1000, followed by incubationwith the substrate solution 3,3′-diaminobenzidine (Sigma). One positiveclone was chosen to continue the experiment.

Testing antibodies—CT was added to sample buffer (3% (w/v) SDS and 5%,(v/v), mercaptoethanol) and electrophoresed in 12% polyacrylamide slabgels, using a discontinuous SDS gel system (Bio Rad). The CT waselectrotransferred onto a Hybond C nitrocellulose filter using asemi-dry system (Bio Rad), and the filter was incubated for at least, 1h, in milk buffer.

The filter was cut into 5-mm wide strips and then incubated separatelyfor 1 h in the relevant sera diluted 1:200 in dilution buffer (PBS with0.05% BSA). After several washes in PBS, the filters were incubated withrabbit anti-chicken IgG-peroxidase conjugate (Sigma) diluted 1:1000 indilution buffer, followed by incubation in the substrate solution3,3′-diaminobenzidine.

(v) Protein and Antibody Assays—Functional Assay—ELISA

Recombinant protein and antibodies produced in response to vaccinationwith yrLTB were determinated by ELISA (enzyme-linked immunosorbentassay).

Testing the ability of the recombinant protein to bind to GM 1 receptor.Each of the following steps was followed by three washes in PBS bufferwith 0.05% Tween 20 and drying on a paper towel.

ELISA plates were incubated for 2 h at 37° C. or overnight at 4° C. withGM1 receptor (monosialoganglioside-GM1, Sigma) diluted incarbonate-coating buffer (pH 9.6) to a final concentration of 15 μg/ml.Skim milk in PBS (1:1, v/v) was added for 1 h at 37° C. as a blockingstep. The supernatant being tested was incubated for 1 h at 37° C. andrabbit anti-CT antibody diluted 1:1000 in PBS buffer was used to detectthe protein. This was followed by incubation with a secondary antibody,goat anti-rabbit IgG conjugated to HRP diluted 1:1000 in PBS buffer. Asubstrate solution, o-phenylenediamine dihydrochloride (Sigma), wasadded and OD was determined by ELISA READER (Lumitron) at 450 nm.

Test recognition of CT by anti-rLTB. Each of the following steps wasfollowed by three washes in PBS buffer with 0.05% Tween 20 and drying ona paper towel.

ELISA plates were incubated for 2 h at 37° C. or overnight at 4° C. withGM1 receptor diluted in carbonate-coating buffer (pH 9.6) to a finalconcentration of 15 μg/ml. Skim milk (50%) in PBS was added for 1 h at37° C. as a blocking step. Commercial CT (Sigma) diluted 1:1000 in PBSbuffer and used as an antigen, was bound to the GM1 receptor for atleast 1 h, at 37° C. The sera being tested was diluted 1:500 in PBSbuffer and incubated for 2 h at 37° C. This was followed by incubationwith a secondary antibody, rabbit anti-chicken IgG-conjugated HRPdiluted 1:1000 in PBS buffer o-Phenylenediamine dihydrochloride wasadded and OD was determined by ELISA READER at 450 nm.

(vi) Optimizing Protein Expression.

The selected clone was grown in 150 ml of BMGY to an OD₆₀₀ of 10 to 20.The culture was centrifuged for 10 min at 6000 RPM. The harvested cellswere resuspended in 30 ml of BMMY, divided into three tubes, 10 ml pertube, and grown for 7 days. LTB expression was determined by adding ofmethanol at concentrations of 0.3, 0.6 and 1.5% twice daily. On days 5,6 and 7, samples of induction medium were collected. A dot blot was usedto detect LTB protein using rabbit anti-CT antibody and goatanti-rabbit-HRP antibody.

(vii) Purification and Concentration of Yeast rLTB Protein.

Ion-exchange chromatography was used for purification of the yrLTBprotein. A strong cation-exchange resin, Macro-Prep High S Support (BioRad), in AKTA prime device (Amersham Pharmacia Biotech), was used forrLTB purification. To adsorb yrLTB to the resin, the induction mediumwith the protein was diluted 1:10 in DDW. Following adsorption of theprotein, the column was washed with 10 volumes of binding buffer (25 mMsodium phosphate, pH 6.8). rLTB was eluted in a linear NaCl gradientwith increasing additions of elution buffer (25 mM sodium phosphate, 1 MNaCl, pH 6.8). yrLTB concentration in the eluates was calculated fromthe BSA curve produced by Bradford test.

(viii) Cloning of the LTB-VP2 Fusion Gene in P. pastoris and Expressionof the Recombinant Protein

The 5′ terminus of the VP2 gene was genetically fused to the 3′ terminusof the LTB gene. The fusion gene LTB-VP2 was constructed by three-stepPCR. A seven-amino-acid, proline-containing linker (Clements. 1990) wasincluded between the LTB and VP2 moieties. The DNA sequences offragments encoding LTB and VP2 were isolated by the two first PCR stepsusing primers of the 5′ and 3′ ends of each gene (GenBank accessionnumbers AB011677 and L42284, respectively). The reactions were performedas described earlier (Materials and Methods, section i).

5′ primer for the VP2+5′Linker construct:

(SEQ ID NO: 3) 5′-gat ccc cgg gta ccg agc tcg aca aac ctg caa gat-3′,

3′ primer for the VP2+5′Linker construct:

(SEQ ID NO: 4) 5′-ccc gga att ctc gag tta cgg cac agc tat cct cct-3′,

5′primer for LTB+3′Linker construct 5′-cga gaa ttc atg gct ccc cag tctatt aca g-3′ (SEQ ID NO: 5), 3′ primer for the LTB1+3′Linker construct5′-cga gct cgg tac ccg ggg atc gtt ttc cat act gat tgc cgc-3′ (SEQ IDNO: 6).

The DNA fragments were purified with GibcoBRL's “PCR productspurification system” and used as a template for synthesis of the fullfusion protein in the third PCR step (with primers of SEQ ID NO: 3 andSEQ ID NO: 6). The PCR scheme was as described earlier (Materials andMethods, section i). The PCR products were electrophoresed in an agarosegel and visualized by ethidium bromide staining.

Following purification with the same kit, the fusion DNA fragment wascloned into the P. pastoris plasmid pHILD2 (Invitrogen) usingrestriction sites incorporated into the primers. The recombinant plasmidwas transformed into Top10 E. coli cells according to the manufacturer'sinstructions. Screening of bacterial colonies was performed as describedpreviously, and the NotI-linearized r-plasmid was cloned into P.pastoris GS1168 according to kit instructions.

Yeast colonies expressing LTB-VP2 protein were screened as described inPitcovski et al. [submitted to Vaccine Journal, 42]. Briefly, the yeastcolonies were placed on a Hybond-C nitrocellulose membrane and grown onMD plates for 2 days at 30° C., and then transferred with the membraneonto MM plates. Following 5 days of induction, the yeast colonies werelysed by yeast lytic enzyme (ICN, Costa Mesa, Calif., USA) and expressedprotein was recognized by anti-CT or anti-IBDV (ABIC, Jerusalem, Israel)antibodies.

The MGY medium was inoculated with the selected colony and incubated at30° C. to an OD₆₀₀ of 1 to 2. The culture was centrifuged andresuspended in MM medium. rLTB-VP2 production was induced by theaddition of methanol every 12 h for 5 days. Following induction, thecells were broken by vortexing with glass beads, centrifuged, and thesupernatant contained the soluble rLTB-VP2. Supernatant was analyzed bySDS-PAGE and immunoblot using anti-CT antibody or polyclonal anti-IBDVantibody.

(ix) Immunogenicity of Recombinant LTB Protein in Chickens

Two sets of experiments were carried out, one on laying hens andbroilers, and the second on one-day-old chicks. Six groups of six birdseach were included in each experimental trial. Birds were treated twiceat a 3-week interval. Birds were vaccinated intramuscularly or orallywith 3 μg of rLTB. CT (50 μg) was used as a positive control and thesupernatant from wild-type plasmid was used as a negative control. Noadjuvant was added to the experimental vaccines. Blood was drawn 3 weeksafter the second vaccination and sera were kept at −20° C. until use.The presence of antibodies in the sera was tested by Western blottingand ELISA, as described previously.

The second set of experiments was comprised of four groups with fivechicks in each. Three groups were vaccinated orally, intramuscularly orby eye-drops with 17 μg of purified yrLTB, without adjuvant. The fourthuntreated group served as a negative control. Blood was drawn two weekspost vaccination and sera were stored at −20° C. until use. Antibodylevels against LT were tested by ELISA as previously described.

(x) Immunogenicity of Recombinant LTB-VP2 Protein in Chickens

Two experiments were conducted to test rLTB-VP2. In one experiment, fivegroups of 10 birds each were tested. Birds, at the age of 5 weeks, werevaccinated twice at a 3-week interval intramuscularly with 150 or 30 μgof rLTB-VP2, or orally with 150 μg of rLTB-VP2. The commercial vaccine(Bursative 2, ABIC) against IBDV, containing VP2 in adjuvant, was usedas a positive control and lysate of wild-type plasmid was used as anegative control. Blood was drawn 3 weeks after each vaccination. Thepresence of antibody was tested by agar gel precipitation (AGP) andELISA using CT and IBDV as antigens as described previously. IBDVchallenge was performed 3 weeks after the second vaccination aspreviously described (Pitcovski et al., 1996).

In the second experiment, three groups of three chicks each werevaccinated via eye-drops with 50 μg yrLTB in 1001 sodium phosphatebuffer, or 50 μg of LTB-VP2 fusion protein in 2001 sodium phosphatebuffer, or VP2 commercial vaccine as a positive control. No adjuvant wasadded to the experimental vaccines. Blood was drawn 2 weeks aftervaccination and sera were stored at −20° C. until use. Antibody levelsin the sera were tested by ELISA using CT and VP2 as antigens, asdescribed previously.

Example 1 Extraction of the Plasmid from E. coli and Cloning the LTBGene in the Yeast

Three plasmids were extracted from E. coli. H10407 (Inoue et al., 1993).Two bands were identified in the agarose gel, the smaller one carryingthe LTB gene.

The open reading frame of LTB from a plasmid extracted from E. coli wasamplified by PCR using oligonucleotides corresponding to both ends ofthe desired gene, as described in Materials and Methods. The PCR productwas electrophoresed in a 1% agarose gel and visualized byethidium-bromide staining (FIG. 1). One sharp band of amplified LTB DNAcould be seen at the expected size (approximately 310 bp). The DNAfragment was extracted and cloned into E. coli, and PCR, restrictionanalysis and DNA sequencing confirmed correct cloning of the LTB.Following amplification of the recombinant plasmid, the construct wascloned into yeast cells.

Example 2 Screening for Pichia pastoris Colonies Expressing LTB

Following 7 days of methanol induction, the nitrocellulose filtercarrying the yeast-colony proteins was probed with specific antibodies(FIG. 2). The screening method for expressing yeast colonies, which wasdeveloped in the laboratory of the present inventors, allows directidentification of colonies expressing the desired protein. A clearlyvisible circle appeared in some of the colonies (19, 29, 33, 35, 36, 38and 39) but not in the negative control (colonies 50 and 51). About 15%of the colonies were found to express yrLTB.

Example 3 Expression and Purification of yrLTB in Yeast Culture

rLTB production was induced in all positive colonies (1 ml culture) andscreened for yield. The colony yielding the highest protein expressionwas chosen for further experiments. Supernatant samples of the selectedcolony were collected on days 5, 6 and 7 of incubation and analyzed bySDS-PAGE (FIG. 3A) and Western blotting (FIG. 3B). Pentameric yrLTB wasseen on all days and identified by specific antibodies against CT (lanes5-7). No bands were found in the negative control.

The expressed protein was tested by ELISA and found to bind to theganglioside receptor GM1. The results shown in Table 1 indicate that LTBwas in the correct pentameric form. Boiling yrLTB for 5 to 10 min causeddenaturation of the pentameric structure and almost completely abolishedGM1 binding.

TABLE 1 Results of an ELISA testing for rLTB's ability to bind theganglioside receptor GM1. OD_(450 nm) Yeast WT yeast Bacterial WT yeastAntigen rLTB plasmid rLTB plasmid ELISA controls 37° C. 1.53 0.28 1.580.30 CT as an 1.66 antigen Boiled 0.16 0.14 0.33 0.26 without Ag 0.26without AbI 0.05 WT—wild type; CT—cholera toxin; Ag - antigen; AbI -rabbit anti-CT antibody

While attempting to improve yrLTB expression, we found that the level ofexpression is proportional to the methanol concentration during theinduction (FIG. 4). Each sample (70 μl) was loaded onto a nitrocellulosemembrane and exposed to specific antibodies. The maximal yield of yrLTBwas obtained at 7 days' induction with 1.5% methanol. These optimalconditions were used for large-volume induction of yrLTB protein forsubsequent in vivo experiments.

Example 4 Purification and Concentration of Yeast rLTB Protein

The pentameric LTB protein is a strong cation. yrLTB was purified bycation-exchange chromatography (FIG. 5). Binding to the resin wasperformed under neutral pH conditions and elution was affected by a NaClcontinuous gradient. SDS-PAGE (FIG. 6A) and Western blotting (FIG. 6B)confirmed the purification. A strong band of pentameric yrLTB, ormonomeric yrLTB after boiling, could be seen in the elution fraction(lane 4 and lane 8, respectively).

To confirm these results, fractions from different point in theseparation process were tested by ELISA for their ability to bind to GM1receptor. The results are shown in Table 2. No rLTB was identified inthe wash fraction, but high titers were identified in three of theeluted fractions.

TABLE 2 ELISA to test the ability of purified rLTB proteins to bindganglioside receptor GM1. Fractions Fractions before transferredseparation column Elution fractions OD_(450 nm) OD_(450 nm) OD_(450 nm)wt wt wt RLTB plasmid rLTB plasmid rLTB % NaCl plasmid 1.19 0.11 0.290.12 0.09 38% 0.10 Commercial 0.26 0.12 1.11 42% 0.15 CT-1.50 without0.24 0.09 1.21 49% 0.14 AbI-0.08 0.23 0.09 1.22 54% 0.08 0.78 60% 0.100.38 66% 0.08

Example 5 Expressing yrLTB-VP2 Fusion Protein in P. pastoris

The DNA fragments of LTB and VP2 with the proline linker at the 3′ and5′ terminus, respectively, were amplified by PCR. The PCR products (FIG.7, FIG. 8) were used as templates to synthesize the fusion gene. The DNAfragment was cloned into P. Pastoris plasmid. The recombinant plasmidwas transformed into E. coli, followed by cloning into P. pastoris hostcells.

Expression of the recombinant protein in P. pastoris was induced bymethanol and the cells were disrupted by glass beads and centrifuged.yrLTB-VP2 was found in the supernatant and analyzed by SDS-PAGE (FIG.9A) and Western blot (FIG. 9B). Both pentameric and monomeric forms ofthe yrLTB-VP2 fusion protein appeared at the expected sizes. The boiled,denatured recombinant protein was recognized by anti-CT (FIG. 7B) andanti-IBDV antibodies (data not shown).

The expressed protein was tested by ELISA and found to bind GM1 (Table3). It should be pointed out that yrLTB was in the correct pentamericstructure, and fusion of a foreign protein to its 3′ terminus did notchange its folding. The native form of yrLTB-VP2 was recognized byanti-IBDV, but not by anti-CT. Recognition by the former indicatescorrect folding of the VP2 protein.

TABLE 3 Results of ELISA testing the ability of LTB-VP2 to bind theganglioside receptor GM1. Anti-CT antibodies Anti-IBDV antibodies LTB-WT Commercial LTB- WT Commercial OD_(450 nm) VP2 plasmid CT VP2 plasmidCT 37° C. 0.38 0.52 1.82 1.21 0.60 0.60 Boiled 0.40 0.44 0.6 0.67 0.660.68 Without 0.46 0.60 Ag Without 0.35 0.60 AbI WT—wild type; CT—choleratoxin; Ag - antigen; AbI - rabbit anti-CT antibody.

Example 6 Immunogenicity of Recombinant LTB Protein in Chickens

The expressed yrLTB protein was injected intramuscularly or administeredorally to broilers. Blood was taken 3 weeks after the secondvaccination. The ability of yrLTB to elicit an immune response and toinduce antibody secretion was tested by Western blotting (FIG. 10) andELISA (FIG. 11). According to the ELISA results, all six injected birdsand five of their orally vaccinated counterparts produced antibodiesthat recognized commercial CT.

The laying hens showed a similar response to the vaccination. Thedifference in antibody level between the experimental group and thenegative control was significant.

One-day-old birds vaccinated with yrLTB, orally or via eye-drops, showedan antibody response. No antibodies were detected by ELISA in birdsvaccinated intramuscularly (FIG. 12).

Example 7 The Adjuvant Effect of yrLTB Protein in Turkeys

The ability of yrLTB to increase the response against the Pasteurellamultocida type 3 (Pm3) vaccine was tested. The experiment included threegroups, 14 turkeys per group, which were vaccinated twice at a 3-weekinterval, followed by challenge with pathogenic P. multocida bacteria(95 cfu per poult). The tested groups were intramuscularly injected with0.05 ml of inactivated Pm3 in emulsion and 2 to 3 μg yrLTB. Pm3 bacteriain commercial water-in-oil adjuvant was used as a positive control andthe PBS buffer was used as a negative control. The rLTB wasintramuscularly injected as an adjuvant for fowl cholera (“cholerin”)vaccine. The ability of the recombinant protein to increase the responseto the vaccine was tested by challenging the birds with pathogenicbacteria. The results are shown in Table 4. The addition of rLTBincreased the response by 14% relative to negative-treated birds, to thesame protection level as the positive control.

TABLE 4 The adjuvant effect of rLTB in protection against virulentcholerin following two vaccinations. Type of vaccination % Hedelston %of post-challenge death Pm3 + rLTB + w/o 93 1/14 PBS + rLTB + w/o 793/14 Positive control 93 1/13

% Hedelston is an indicator for protection against cholerin. Up to 60%is regarded as positive response.

Example 8 Immunogenicity of yrLTB-VP2 Protein in Chickens

The fusion yrLTB-VP protein was injected intramuscularly or administeredorally to birds without additional adjuvant. The ability of yrLTB-VP2 toinduce antibodies and to protect chickens against IBD challenge isdemonstrated in FIG. 13 and Table 5. No antibodies against LTB werefound. After the first vaccination, ELISA with IBDV as the antigenshowed that chickens injected with 150 μg of fusion protein developed ahigh level of antibody. Following the second vaccination, both 150 and30 μg injected groups developed anti-IBDV antibodies. In the groupinjected with a 150 μg of yrLTB-VP2 protein, all the birds resistedchallenge with virulent virus, while in the group injected with 30 μg, 7of 10 birds showed full resistance, while in the control group, non ofthe birds were protected. In the orally vaccinated group, the efficacyof the recombinant protein was partial—only 22% of the birds wereresistant to challenge.

TABLE 5 Efficiency of rLTB-VP2 in protection against virulent IBDVfollowing two vaccinations. Type of vaccination % Resistance tochallenge Injection of 150 μg rLTB-VP2 100%  Injection of 30 μg rLTB-VP270% Oral administration of 150 μg rLTB-VP2 22% Injection of wt plasmid 0% Commercial vaccine 100% 

In response to vaccination with LTB-VP2 fusion protein by eye-drops, thechicks produced high levels of anti-VP2, but no anti-CT antibodies,whereas birds vaccinated with rLTB exhibited high titers of anti-CTantibodies (Table 6).

TABLE 6 The antibody response in chicks eye-drop-vaccinated with rLTB,VP2 or rLTB-VP2 fusion protein at two weeks of age. Ag in ELISA VaccineLTB VP2 LTB 0.63 — LTB-VP2 0.14 0.63 VP2 — 0.37 Ag—antigen

Example 9 Cloning the LT Gene in the Yeast Pichia pastoris

The plasmid from E. coli H10407 (Yamamoto and Yokota, 1983 vol. 153;Inoue, 1993) is used as a template to synthesize the LT fragment. TheDNA sequence of the fragment encoding the LT is propagated by PCR usingprimers of the ends of the gene as published previously (GenBankaccession number AB011677).

The reaction solution contains 1 unit of Taq polymerase (Promega,Madison, Wis., USA), 5 μl of Taq buffer (20 mM Tris-HCl pH 8.3, 50 mMKCl, 2 mM MgCl₂) and 20 pmol of each primer in a final volume of 50 μl.The PCR scheme was as follows: 5 min at 94° C., 2 min at 42° C., 60 s at74° C., (60 s at 94° C., 2 min at 50° C., 3 min at 74° C.)×28 cycles, 15min at 74° C. The PCR product was electrophoresed in a 1% agarose geland visualized by ethidium bromide staining.

The DNA fragment is purified with a “PCR products purification system”kit (GibcoBRL, UK) and cloned into a pHILSI plasmid (Invitrogen, SanDiego, Calif., using the XhoI and BamHI restriction sites incorporatedinto the primers. The recombinant plasmid is transformed into Top10 E.coli cells (Invitrogen) according to the manufacturer's instructions.Screening of bacterial colonies, cloning into P. pastoris GS1168 andscreening of yeast colonies expressing LT protein are performed asdescribed in Materials and Methods above.

Example 10 Cloning the LTA Gene in the Yeast Pichia pastoris

The plasmid from E. coli H10407 (Yamamoto and Yokota, 1983 vol. 153;Inoue, 1993) is used as a template to synthesize the LTA fragment. TheDNA sequence of the fragment encoding the LTA is propagated by PCR usingprimers of the ends of the gene as published previously (GenBankaccession number AB011677).

The reaction solution contains 1 unit of Taq polymerase (Promega,Madison, Wis., USA), 5 μl of Taq buffer (20 mM Tris-HCl pH 8.3, 50 mMKCl, 2 mM MgCl₂) and 20 pmol of each primer in a final volume of 50 μl.The PCR scheme was as follows: 5 min at 94° C., 2 min at 42° C., 60 s at74° C., (60 s at 94° C., 2 min at 50° C., 3 min at 74° C.)×28 cycles, 15min at 74° C. The PCR product was electrophoresed in a 1% agarose geland visualized by ethidium bromide staining.

The DNA fragment is purified with a “PCR products purification system”kit (GibcoBRL, UK) and cloned into a pHILSI plasmid (Invitrogen, SanDiego, Calif., USA) using the XhoI and BamHI restriction sitesincorporated into the primers. The recombinant plasmid is transformedinto Top10 E. coli cells (Invitrogen) according to the manufacturer'sinstructions. Screening of bacterial colonies, cloning into P. pastorisGS1168 and screening of yeast colonies expressing LTA protein areperformed as described in Materials and Methods above.

Example 11 Cloning the CT Gene in the Yeast Pichia pastoris

The gene from Vibrio cholerae O27 is used as a template to synthesizethe CT fragment. The DNA sequence of the fragment encoding the CT ispropagated by PCR using primers of the ends of the gene as publishedpreviously (GenBank accession number AF390572).

The reaction solution contains 1 unit of Taq polymerase (Promega,Madison, Wis., USA), 5 μl of Taq buffer (20 mM Tris-HCl pH 8.3, 50 mMKCl, 2 mM MgCl₂) and 20 pmol of each primer in a final volume of 501.The PCR scheme was as follows: 5 min at 94° C., 2 min at 42° C., 60 s at74° C., (60 s at 94° C., 2 min at 50° C., 3 min at 74° C.)×28 cycles, 15min at 74° C. The PCR product was electrophoresed in a 1% agarose geland visualized by ethidium bromide staining.

The DNA fragment is purified with a “PCR products purification system”kit (GibcoBRL, UK) and cloned into a pHILSI plasmid (Invitrogen, SanDiego, Calif., USA) using the XhoI and BamHI restriction sitesincorporated into the primers. The recombinant plasmid is transformedinto Top10 E. coli cells (Invitrogen) according to the manufacturer'sinstructions. Screening of bacterial colonies, cloning into P. pastorisGS1168 and screening of yeast colonies expressing CT protein areperformed as described in Materials and Methods above.

Example 12 Cloning the CTB Gene in the Yeast Pichia pastoris

The gene from Vibrio cholerae O27 is used as a template to synthesizethe CTB fragment. The DNA sequence of the fragment encoding the CTB ispropagated by PCR using primers of the ends of the gene as publishedpreviously (GenBank accession number AF390572 (only CTB-U25679).

The reaction solution contains 1 unit of Taq polymerase (Promega,Madison, Wis., USA), 5 μl of Taq buffer (20 mM Tris-HCl pH 8.3, 50 mMKCl, 2 mM MgCl₂) and 20 pmol of each primer in a final volume of 50 μl.The PCR scheme was as follows: 5 min at 94° C., 2 min at 42° C., 60 s at74° C., (60 s at 94° C., 2 min at 50° C., 3 min at 74° C.)×28 cycles, 15min at 74° C. The PCR product was electrophoresed in a 1% agarose geland visualized by ethidium bromide staining.

The DNA fragment is purified with a “PCR products purification system”kit (GibcoBRL, UK) and cloned into a pHILSI plasmid (Invitrogen, SanDiego, Calif., USA) using the XhoI and BamHI restriction sitesincorporated into the primers. The recombinant plasmid is transformedinto Top10 E. coli cells (Invitrogen) according to the manufacturer'sinstructions. Screening of bacterial colonies, cloning into P. pastorisGS1168 and screening of yeast colonies expressing CTB protein areperformed as described in Materials and Methods above.

Example 13 Cloning the CTA Gene in the Yeast Pichia pastoris

The gene from Vibrio cholerae 027 is used as a template to synthesizethe CTA fragment. The DNA sequence of the fragment encoding the CTA ispropagated by PCR using primers of the ends of the gene as publishedpreviously (GenBank accession number AF390572 (only CTA-A16422).

The reaction solution contains 1 unit of Taq polymerase (Promega,Madison, Wis., USA), 5 μl of Taq buffer (20 mM Tris-HCl pH 8.3, 50 mMKCl, 2 mM MgCl₂) and 20 pmol of each primer in a final volume of 501.The PCR scheme was as follows: 5 min at 94° C., 2 min at 42° C., 60 s at74° C., (60 s at 94° C., 2 min at 50° C., 3 min at 74° C.)×28 cycles, 15min at 74° C. The PCR product was electrophoresed in a 1% agarose geland visualized by ethidium bromide staining.

The DNA fragment is purified with a “PCR products purification system”kit (GibcoBRL, UK) and cloned into a pHILSI plasmid (Invitrogen, SanDiego, Calif., USA) using the XhoI and BamHI restriction sitesincorporated into the primers. The recombinant plasmid is transformedinto Top10 E. coli cells (Invitrogen) according to the manufacturer'sinstructions. Screening of bacterial colonies, cloning into P. pastorisGS1168 and screening of yeast colonies expressing CTA protein areperformed as described in Materials and Methods above.

Example 14 Expression of LT, CT, LTB and CTB in CHO Cells

The expression constructs are created by using pCI-neo (Promega) and thePCR products of LT, CT, LTB and CTB. The K1 line of CHO cells isobtained from the American Type Culture Collection (Manassas, Va.). Thecells are grown in RPMI medium 1640 (Life Technologies, Gaithersburg,Md.) supplement with 10% heat-inactivated FCS (Life Technologies), 20 mMHepes (pH 7.2; Life Technologies), 4 mM L-glutamine (Gibco-BRL) andpenicillin/streptomycin (Gibco-BRL). Cells are transfected with 2.5 μgof expression vectors, or empty vector by using the Superfecttransfection reagent (Qiagen) according to the manufacturer'srecommendations, and selected with 1 mg/ml Geneticin (LifeTechnologies). Stable transfectants of CHO K1 cells are selected. Theexpression of LT, CT, LTB and CTB in the selected clones is tested byWestern blot analysis using anti-CT or anti-LT antibodies (ABIC,Jerusalem, Israel) as described in Materials and Methods above.

Example 15 Expression of LT, CT, LTB and CTB in Insect Cells

The PCR products of LT, CT, LTB and CTB are digested with EcoRI and thenligated into the EcoRI site of the baculovirus transfer vector pBacPAK8(Clontech, Palo Alto, Calif.). High Five (HF) cells infected with therecombinant baculovirus (10 PFU/cell) are incubated with 1 ml of aprotein-free Sf-900 II SFM medium (Gibco BRL, Rockville, Md.) for 4days. After incubation, the cells and culture medium mixtures arecentrifuged at 1,400×g for 5 min at 4° C., and the supernatants arefurther centrifuged at 99,000×g for 2 h at 4° C. to get rid of theviruses. The resulting supernatants are collected and used for furtherexperiments. The infected cells are washed twice with PBS bycentrifugation at 5,000 rpm for 5 min at 4° C., and then resuspended in1 ml of PBS for further analysis. The infected cells and thesupernatants are mixed with an equal volume of 2× sodium dodecyl sulfate(SDS) gel-loading buffer (100 mM Tris-HCl, pH 6.8, 100 mM2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol). Thepurified LT, CT, LTB and CTB are mixed with an equal volume of an SDSgel-loading buffer under reducing conditions. The samples are boiled for5 min, and then subjected to Western blot analysis using anti-CT oranti-LT antibodies (ABIC, Jerusalem, Israel) as described in Materialsand Methods above.

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1. A recombinant toxin or the subunit B thereof selected from the groupconsisting of E. coli heat-labile enterotoxin (LT), the subunit B of TL(LTB), cholera toxin (CT) and the subunit B of CT (CTB), in immunogenicform, wherein said immunogenic toxin or the subunit B thereof has beenexpressed in eukaryotic cells.
 2. The recombinant LT, LTB, CT or CTB ofclaim 1, wherein said eukaryotic cells are yeast cells, mammalian cellsor insect cells.
 3. The recombinant LT, LTB, CT or CTB of claim 2,wherein said yeast cells are Pichia pastoris cells, said mammalian cellsare Chinese hamster ovary (CHO) cells, and said insect cells are thehigh five (HF) cell line. 4-7. (canceled)
 8. A vaccine containing therecombinant LT, LTB, CT or CTB of claim
 1. 9. The cholera vaccine ofclaim 8 containing the recombinant CT or CTB.
 10. The vaccine of claim 8against E. coli heat-labile enterotoxin containing the recombinant LT orLTB.
 11. The vaccine of claim 10 containing recombinant LTB expressed inPichia pastoris.
 12. A vaccine comprising the recombinant LT, LTB, CT orCTB of claim 1 and a further antigen, wherein said recombinant LT, LTB,CT or CTB and said antigen are in mixture, chemically linked or form afusion protein. 13-16. (canceled)
 17. The vaccine of claim 12 forpoultry vaccination against infectious bursal disease virus (IBDV)containing recombinant LT, LTB, CT or CTB produced in eukaryotic cellsand the IBDV Viral protein 2 (VP-2) antigen.
 18. The IBDV vaccine ofclaim 17 for poultry vaccination wherein the recombinant LT, LTB, CT orCTB and the IBDV VP-2 antigen form a fusion protein.
 19. The IBDVvaccine of claim 17 for poultry vaccination containing a fusion proteincomprising recombinant LTB produced in yeast and the IBDV VP-2 antigen.20. The IBDV vaccine of claim 19 wherein the LTB and the VP-2 moietiesare linked by a linker peptide.
 21. A recombinant fusion proteincomprising LT, LTB, CT or CTB and a further antigen that has beenexpressed in Pichia pastoris expression system.
 22. The recombinantfusion protein of claim 21, consisting of LTB-VP2. 23-41. (canceled) 42.A vaccine as claimed in claim 8 for oral, intranasal, mucosal, eyedrops, vaginal, rectal, or transcutaneous administration.
 43. A human orveterinary vaccine comprising an adjuvant consisting of recombinant LT,LTB, CT or CTB produced in eukaryotic cells.
 44. A method forvaccination comprising administering a human or veterinary vaccinecomprising an adjuvant consisting of recombinant LT, LTB, CT or CTBproduced in eukaryotic cells, or co-administering said vaccine withrecombinant LT, LTB, CT or CTB produced in eukaryotic cells.
 45. Themethod according to claim 44 wherein the vaccine is a veterinary choleraor Pasteurella multocida vaccine and the adjuvant is LTB.